Studies of mutation rates and molecular spectra at the endogenous hprt gene are underway using human cancer cell lines. These studies address the hypothesis that there exist functional differences among human mismatch repair (MMR) gene products that have specific effects on spontaneous and damage induced mutagenesis. Mutation rates at hprt were measured in human cancer cell lines defective in the MMR genes hMLH1, hPMS2, or GTBP. These MMR-deficient cell lines exhibited a 50-750 fold increase in mutation rate relative to a MMR-proficient cancer cell line. These data support the notion that MMR plays an important role in controlling the rate of spontaneous mutation and suggest that different MMR-gene defects vary in their ability to repair different types of DNA mismatches. Finally, studies are designed toward the goal of providing insight into the cellular pathways leading to mutations in mammalian cells. These studies are intended to further characterize already widely used in vitro mammalian mutagenesis assay systems and to assess the role of specific mismatch repair pathways in the regulation of spontaneous and induced mutation. We have further characterized two cell lines widely used as in vitro mammalian mutagenesis assay systems: mouse L5178Y and Chinese Hamster ovary (CHO) AS52 cells. The gpt integration site in AS52 cells has been cloned and sequenced in order to define DNA sequence motifs involved in contributing to the exquisite sensitivity of this cell line to genomic rearrangements induced by environmental carcinogens. There are numerous direct and inverted repeats that most likely contribute to rearrangements induced by such agents leading to deletions. In addition, we are continuing to characterize the status of the p53 gene in L5178Y cells.